Mast cells are known to be involved in vascular inflammation and atherosclerosis. Increasing evidence also suggests a role for infectious agents in initiating atherogenic events in the vessel wall. Our preliminary results show that histamine and tryptase, two major products of the mast cell, enhances endothelial cell (EC) inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components presumably via upregulation of Toll-like receptor-2 and -4 (TLR2 and TLR4). The long-term objective of this grant application is to delineate the mechanism by which mast cell-derived histamine and tryptase act in synergy with the TLR ligands to amplify vascular inflammation and participate in the progression of atherosclerosis. The specific aims of this project are: 1) Determine whether histamine and tryptase regulate the expression and function of TLR2 and TLR4 in normal human coronary artery endothelial cells (HCAEC). 2) Determine whether histamine-and tryptase-primed normal HCAEC and endothelial cells isolated from human atherosclerotic vessels are phenotypically similar and hyperresponsive to TLR2 and TLR4 ligands. 3) Determine whether histidine decarboxylase knockout mice (HOC-/-) and mast cell-deficient (Sl/SId) mice have decreased endothelial expression of TLR2 and TLR4 and are hyporesponsive to Gram-negative and Gram-positive bacterial PAMPs in vivo and in vitro. 4) Determine whether mast cell degranulation will accelerate the progression of atherosclerosis in an apoE-/- mice infected with C. pneumoniae, and evaluate whether changes in atherosclerotic lesions correlate with the endothelial expression of TLR2 andTLR4. The study will utilize real-time PCR, immunofluorescence and Western blot analyses, flow cytometry, confocal microscopy, and ELISA techniques, as applicable. Upon completion, the proposed studies will reveal the mechanism by which histamine and tryptase upregulate infection-associated vascular inflammation and will enhance our understanding of the role of mast cells in the pathogenesis of atherosclerosis. [unreadable] [unreadable]